The control of Plasmodium falciparum malaria by vaccination will require immunization with multiple parasite antigens effectively formulated in combination. In this regard, proteins expressed on the surface of blood-stage merozoites are attractive as vaccine targets given their functional importance in the invasion of erythrocytes and accessibility to serum antibodies. However, progress toward human trials of merozoite antigen based vaccines has been hampered by an inadequate understanding of protective responses and a lack of measurable correlates of that immunity. The objective of this application is to define parameters of protective immunity targeted by vaccination with two merozoite surface proteins, apical membrane antigen-1 (AMA-1) and merozoite surface protein-1 (MSP-1). Since optimal formulations for immunizations with AMA-1 or MSP-1 alone may differ, our focus will be on formulations that are effective for combined antigen immunizations. We have a simple, workable system using combinations of recombinant AMA-1 and MSP-1 to immunize mice against Plasmodium chabaudi malaria. A good B cell response and significant antibody production are necessary for protection and can be achieved utilizing Alum or Quil A as adjuvant. However, prechallenge antibody titers alone are not predictive of protective efficacy. Furthermore, studies in B cell deficient mice revealed an antibody independent, cell mediated component to Pc MSP-1 induced protection. We will examine antibody specificity, concentration, isotype and avidity to define measurable parameters that predict protective efficacy. By large-scale DNA microarray analyses, we will determine gene expression profiles of CD4+ T cells from Pc AMA-1 + Pc MSP-1 immunized and protected animals compared to those from mice fully susceptible to P. chabaudi malaria. Correlates of Pc AMA-1 + Pc MSP-1 induced protective T cell and B cell responses defined in vitro will be validated in vivo by passive immunization studies and through the immunization of selected immunological knockout mice. We expect that the information gained can be applied to the evaluation of immune responses in Aotus monkeys and humans as combined formulations of AMA-1 and MSP-1 move into clinical trials.